Knockdown of p21^Cip1/Waf1 enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells

Abstract

SummaryMammalian aging of many tissues is associated with a decline in the replicative and functional capacity of somatic stem cells. Understanding the basis of this decline is a major goal of aging research. Human bone marrow-derived multipotent stromal cells (MSCs) have been applied in the treatment of fracture nonunion. Clinical application of MSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often at the expense of stemness and differentiation potentiality. We first demonstrated that late-passage MSCs exhibited decreased proliferation capacity, reduced expression of stemness markers such as Oct-4 and Nanog, and deterioration of osteogenic potential. Further, late-passage MSCs showed increased expression of p21 Cip1 ⁄ Waf1 (p21), an inhibitor of the cyclin-dependent kinase. Knockdown of p21 by lentivirus-mediated shRNAs against p21 in late-passage MSCs increased the proliferation capacity, the expression of Oct-4 and Nanog, and osteogenic potential compared with cells transduced with control shRNA. More importantly, reduction in p21 expression in MSCs enhanced the bone repair capacity of MSCs in a rodent calvarial defect model. Knockdown of p21 in MSCs also increased the telomerase activity and telomere length, and did not show chromosomal abnormalities or acquire transformation ability. Therefore, these data successfully demonstrate the involvement of senescence gene in the expression of stemness markers and osteogenic potential of MSCs.

Increased levels of p21(CIP1/WAF1) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

“…Previous animal studies have implicated p21 (CIP1/WAF1) in tissue repair/regeneration, with p21 deficient mice being able to repair significant trauma to appendages without scarring . Furthermore, at least two studies by independent labs have shown that mesenchymal stem cells deficient in p21 expression have increased potential to differentiate into both adipocytes and osteoblasts (Yew et al, 2011;Cheng et al, 2011). Additionally, the recent study by Yew et al (2011) suggested that p21 levels fluctuated in bone marrow MSCs collected from different patients.…”

“…1D). Since it had been previously reported that p21 levels in MSC populations correlated with osteogenic and adipogenic differentiation capacity (Yew et al, 2011), we next examined if the levels of p21 in the undifferentiated synovial MPCs was related to the chondrogenic differentiation capacity of the cells.…”

“…However, it remains unknown if inflammation within the synovial environment regulates the expression of p21 in these MPCs. A study by Yew et al (2011) examined the role of p21 in bone marrow MPCs, and through an Sh-RNA knock-down approach they were able to demonstrate that decreased levels of p21 resulted in an increased 'stemness' phenotype, telomere length and concurrent increased osteogenic differentiation capacity (in vitro and in vivo). However, one observation in this study that was not discussed was that the three donors examined all had distinct levels of p21 expression before Sh-RNA knock-down.…”

Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

“…Our findings that altering p21 levels affects the expansion rates and GAG production of iPSC-derived chondrocytes are consistent with previous results in human MSCs showing that reduced p21 expression through the use of hypoxia, growth factors, or siRNA can increase both proliferative and differentiation potential. 24,25,38 An important result of p21 silencing was attenuation of the sharp decrease in cell number associated with early stages of pellet culture in control cells, a phenomenon that also occurs in other cell types. 29 BrdU staining indicated that increased DNA synthesis was one factor in the higher DNA content of pellets from p5 p21sh cells compared with controls, but silencing p21 may also have played a role in Images representative of pellets from two independent cell preparations.…”

WW Domain-containing E3 Ubiquitin Protein Ligase 1 (WWP1) Delays Cellular Senescence by Promoting p27Kip1 Degradation in Human Diploid Fibroblasts

“…The double-stranded DNA was inserted into the EcoRI and XhoI sites of the pMSCV-puro-miR30 vector. The following gene-specific sequences were used successfully: WWP1, 5Ј-GAGTTGATGATCGTAGAAG-3Ј (11); p21 Cip1 , 5Ј-GACAGATTTCTATCACTCCAA-3Ј (22). For transient transfection, HeLa cells at 70 -80% confluence were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions.…”